The focus of this project is characterize signal transduction pathways downstream of K-Ras that mediate the pathogenesis of pancreatic ductal adenocarcinoma (PDAC). Our working hypothesis is that K-Rasdependent activation of phosphoinositide 3-kinase (PI3K) plays a critical role in this cancer. We speculate that pathways downstream of PI3K play diverse biological roles at every step along the PDAC progression process?specifically effecting rapid cell proliferation, resistance to apoptosis, aggressive motogenic behavior and angiogenesis. Further we propose that the detailed identification of both the upstream and downstream components of this signaling network would provide information on critical targets for improved therapeutics. The activation state of components of the Ras/PI3K/Akt signaling pathway will be assessed in the various mouse models for multistage PDAC being developed in Project 1. These models include expression of mutant K-Ras in the pancreas in the context of germline loss of various tumor suppressor genes (INK4a/Arf, p53; and/or SMAD4). Phospho-specific antibodies will be utilized in western blots, tissue staining and cytoimmunoflourescence to define the activation state of protein kinases downstream of Ras, including ERK, AKT, tuberin, FOXO family members, p70S6K, S6 and 4EBP1 as a function of tumor progression in the mouse models that are generated and phenotypically characterized in Projects 1 and 4. In addition these same components will be evaluated in human tissues and cell lines available from the Biobank Core. The activation of these pathways will be compared to the genetic profiles determined in Project 1 in order correlate genomic perturbations with biochemical responses. A strong emphasis will be placed on how these activation events relate to increasing grades of PanIN and the tumor biological properties of advanced PDAC tumors including angiogenesis in collaboration with Project 3. This same analysis will be used to characterize the properties of potential PDAC cancer stem cells defined in Project 4. In addition, the requirement for PI3K signaling components for tumorigenic growth in human PDAC cell lines will be evaluated using inducible shRNA vectors. Finally, a rigorous genetic approach to assess the functional role of PI3K signaling in PDAC pathogenesis will be undertaken by crossing mice that are conditionally deficient in PI3K subunits to the aforementioned mouse models. The identification of the crucial mediators of K-Ras-dependent tumorigenesis across the well-defined tumor contexts will illuminate future drug targets and pinpoint the critical molecules underpinning the biological progression of this deadly disease.